Oligonucleotides are widely used in DNA technologies. One of the most important properties of an oligonucleotide is its ability to bind to a complementary sequence in other polynucleotides. Robust and specific annealing of an oligonucleotide to its complementary sequence is important for the success of probe hybridization methods that allow detection and quantification of pathogens, genomic mutations and other nucleotide sequences. Unfortunately, some oligonucleotides composed of the naturally occurring nucleotides cannot be used as robust probes. For example, an oligonucleotide containing two segments of sequences that are complementary to each other (e.g., CAAAAAAAAAACACTTTTTTTTTT (SEQ ID NO: 67)) would form an internal structure called a hairpin that would prevent hybridization to its target. A further example is an oligonucleotide that can form a dimer with its second copy (e.g., ACTGAGACTCTAATCGATTAG (SEQ ID NO: 68)). Thus, there is a need for a method to inhibit the formation of such undesired structures.
Another typically unwanted biological or molecular process is the annealing of an oligonucleotide to non-target sequences in polynucleotides, called non-specific hybridization. This process increases the background signal in probe hybridization that limits the applications of this method and may lead to false positive results. The discrimination between specific and non-specific hybridization is most challenging when polynucleotides contain sequences that are similar to the target sequence. Another challenging situation is when very long polynucleotides (e.g., genomic DNA of 1 million (1 Mb) to 3 billion (3 Gb) base pairs) with a large amount of potential non-specific targets are present. Thus, there is a need for a method to inhibit non-specific hybridization of oligonucleotides.
There is a relatively narrow range of conditions (temperature, concentrations of ions and denaturing reagents) at which an oligonucleotide anneals specifically to its complementary target. These conditions are usually determined by measuring melting temperature (Tm) of a duplex comprising an oligonucleotide and the second oligonucleotide that contains a sequence of bases complementary to the first oligonucleotide. Unfortunately, the range of conditions for the specific annealing of an oligonucleotide may not coincide with other requirements of the intended method. A common practice to meet these requirements is to select the length and GC content of an oligonucleotide probe with appropriate melting temperature. This selection may contradict other requirements on the length of an oligonucleotide. For example, a 40-mer oligonucleotide that has only one complementary sequence in a genomic DNA sequence generally has too high of a melting temperature and would anneal to partially complementary targets while a 15-mer oligonucleotide that has a suitable melting temperature would have too many complementary sequences in a genomic DNA sequence. Thus, there is a need for a method to inhibit non-specific hybridization of oligonucleotides at the wide variety of stringency conditions dictated by the requirements, other than the melting temperature, of nucleotide sequences.
Oligonucleotides and complexes with other polynucleotides are widely used as substrates for protein binding and enzymatic reactions. The enzymatic reaction typically results in chemical modification of an oligonucleotide, including cleavage of the oligonucleotide or addition of extra nucleotide(s). The latter reaction may be catalyzed by polymerase that uses an oligonucleotide as a primer and adds bases complementary to the bases in the template polynucleotide. Polymerase may also use an oligonucleotide as a template for polymerization reaction. Enzymatic reactions involving oligonucleotides constitute the core of many DNA technologies, for example, PCR, DNA sequencing, and SNP detection. The formation of undesired structures by an oligonucleotide or its complexes with other polynucleotides may interfere with the intended enzymatic reaction. Moreover, even transient formation of such undesired structures in a minute fraction of oligonucleotides could be amplified by the enzymatic reaction. One example of such an undesired process is the non-specific amplification by PCR that is difficult to avoid if the number of amplification cycles exceeds 40. Another such example is primer-dimer amplification during PCR. Thus, there is a need for a method to inhibit the ability of oligonucleotides to form such undesired structures in enzymatic reactions.
Oligonucleotides may serve different functions in DNA technologies that involve enzymatic reactions. One example is as a probe for detection of specific sequences amplified by PCR with two primers in a TaqMan assay. Such a probe should specifically bind to its complementary sequence and potential polymerization of the probe should be inhibited. Thus, under such circumstances, there is a need for a method to inhibit non-specific hybridization of an oligonucleotide and to inhibit its ability to function as a primer.
Oligonucleotides are also used as primers in primer extension reactions for SNP detection, which comprises one or more cycles of adding, by action of DNA polymerase, a labeled nucleotide to a primer annealed to its target complementary sequence. The results of this method would be jeopardized if the primer extension occurs at sites of non-specific annealing of the primer or if the primer itself serves as a template. For example, a hairpin CAAAAAAAAAACACTTTTTTTTTT (SEQ ID NO: 67) and dimer of ACTGAGACTCTAATCGATTAG (SEQ ID NO: 68) oligonucleotides could serve as templates and the resulting undesired products will be CAAAAAAAAAACACTTTTTTTTTTg (SEQ ID NO: 69) and ACTGAGACTCTAATCGATTAGa (SEQ ID NO: 70).
Oligonucleotides are also used as primers in primer extension reactions for DNA sequencing, which comprises one or more cycles of adding nucleotides, by action of DNA polymerase, to a primer annealed to its target complementary sequence and terminating the extension reaction at a specific base encoded in the template. The undesired processes described in the previous paragraph would jeopardize the results of this method. Undesired primer extension products may have additional bases at their 3xe2x80x2 ends and potentially could prime the reaction from targets that are complementary to the newly formed primers rather than to the original primers. In addition, polynucleotide products generated by the original primer extension could serve as templates for the annealing of the second copy of the primer and its subsequent extension. Should this event occur, it could generate a polynucleotide that has a primer sequence at its 5xe2x80x2 end and a sequence complementary to the primer at its 3xe2x80x2 end. DNA polymerase would generate the latter sequence at the final steps of the extension of the second copy of the primer when the nucleotides that comprise the first copy of the primer serve as templates. This polynucleotide would trigger exponential amplification (non-specific PCR) in a cycle sequencing method based on linear multiplication of products. Eventually, non-specific exponential amplification would overwhelm the linear multiplication and jeopardize the outcome of DNA sequencing. This undesired process limits the utility of such a cycle sequencing method. Thus, there is a need for a method to inhibit non-specific hybridization of an oligonucleotide, while retaining its ability to function as a primer and inhibiting its ability to function as a template in a polymerization reaction.
Oligonucleotides are also used as primers in primer extension reactions for PCR amplification, which comprises several cycles of adding nucleotides, by action of DNA polymerase, to primers annealed to target complementary sequences and termination of the extension reaction at the template end that is composed of nucleotides of another primer. The final 3xe2x80x2 end nucleotide added by DNA polymerase is complementary to the 5xe2x80x2 nucleotide of the other primer, and the final PCR product is the double stranded DNA with blunt ends. However, some polymerases (e.g., Taq polymerase) could add one more non-templated nucleotide (dA). The result would be a mixture of duplexes that differ in length by one nucleotide. This problem makes it difficult to interpret the results of PCR for genotyping. Thus, there is a need for a method to inhibit non-specific hybridization of an oligonucleotide, while retaining its ability to function as a primer for PCR amplification and allowing termination of polymerization reactions at a defined site on the primer when it serves as a template.
Oligonucleotides may serve different functions in DNA technologies. These differences often preclude the use of the same oligonucleotide in different applications. It would be useful to find a method that will allow multiple functions of an oligonucleotide, e.g., its functioning as a primer under one set of conditions and the inhibition of its ability to prime an extension reaction under another set of conditions.
The present invention provides a method for inhibiting undesired molecular interaction between oligonucleotides and their complexes with polynucleotides and enzymes, including local interactions between their chemical units (nucleotides, amino acids).
The present invention provides a method of inhibiting at least one molecular process in a sample, comprising administering to the sample an oligonucleotide or polynucleotide containing at least one monomeric unit having formula (I):
Axe2x80x94Xnxe2x80x83xe2x80x83(I)
wherein A is an organic moiety, n is at least 1, and each X is independently selected from the group consisting of xe2x80x94NRCOCONu, xe2x80x94NHCOCR2CR2CONu, xe2x80x94NHCOCRxe2x95x90CRCONu, and xe2x80x94NHCOSSCONu, wherein each R independently represents H or a substituted or unsubstituted alkyl group, and Nu represents a nucleophile, or a salt of the compound.
The present invention provides a method of inhibiting at least one molecular process in a sample, comprising administering to the sample an oligonucleotide or polynucleotide containing at least one monomeric unit having formula (I):
Axe2x80x94Xnxe2x80x83xe2x80x83(I)
wherein A is an organic moiety, n is at least 1, and each X is independently selected from the group consisting of xe2x80x94NRCOCONu, xe2x80x94NHCOCR2CR2CONu, xe2x80x94NHCOCRxe2x95x90CRCONu, and xe2x80x94NHCOSSCONu, wherein each R independently represents H or a substituted or unsubstituted alkyl group, and Nu represents a nucleophile, or a salt of the compound.
As used herein, oligonucleotide or polynucleotide refers to a macromolecule consisting of a nucleotide chain, which may be of various lengths, and may contain modifications or substitutions at monomeric units of the chain. Such modifications or substitutions should not exceed 50% of the oligonucleotide or polynucleotide.
In embodiments, the oligonucleotide or polynucleotide of the present invention may be modified or substituted at from 1-20 bases, 1-10 bases, such as 1-5 bases, for example 1-2 bases.
Group X may be in various quantities such as 1, 2, 3 ,4, 5, 6, 7, 8, 9, 10, 11, 12 or more such groups. In embodiments, multiple X groups is preferable.
Nucleosides, nucleotides and modified nucleosides and nucleotides may be used as organic moieties in the present invention. Non-nucleosides or non-nucleotides may also be used as organic moieties in the present invention. Suitable non-nucleosides or non-nucleotides of the present invention include, but are not limited to, a substituted or unsubstituted alkane, such as an alkane having from 3 to 100 carbon atoms, preferably from 3 to 20 carbon atoms and more preferably from 3 to 12 carbon atoms; a substituted or unsubstituted cycloalkane, such as a cycloalkane having from 3 to 12 carbon atoms in a cycle, preferably from 4 to 8 carbon atoms in a cycle and more preferably from 5 to 6 carbon atoms in a cycle; and a substituted or unsubstituted heterocyclic compound, such as a heterocyclic compound having from 3 to 20 carbon atoms in a cycle, preferably from 3-14 carbon atoms in a cycle. The compound may be substituted with at least one substituent, such as substituents selected from the group consisting of a hydroxy group, a protected hydroxy group and a halogen.
In embodiments of the invention, the nucleophile is selected from the group consisting of compounds having an xe2x80x94Oxe2x88x92, an amino group (xe2x80x94NH2), a primary amino group (xe2x80x94NRH) and a secondary amino group (xe2x80x94NR2). Suitable nucleophiles are, for example, listed in Table AA herein.
In embodiments, R may be a substituted or unsubstituted alkyl group. The alkyl group may preferably have from 1 to 15, more preferably from 1 to 12, and even more preferably from 1 to 6 carbon atoms.
Other suitable compounds and methods of synthesizing such compounds are disclosed in U.S. Pat. No. 5,902,879 to Polouchine; U.S. patent application Ser. No. 09,655,317, filed Sep. 5, 2000, to Polouchine; and U.S. patent application Ser. No. 09/655,316, filed Sep. 5, 2000, to Polouchine, the entire disclosures of which are hereby incorporated by reference.
In particular, suitable monomers include compounds of the formula II: 
wherein B is purine or pyrimidine moiety, and each Y independently represents H, a group that protects a hydroxy group, a (PO3)mxe2x88x922 group wherein m is an integer of 1-3, a group reactive to link hydroxy groups, or a phosphodiester linkage to another monomer of said oligonucleotide or polynucleotide, and X is selected from the group consisting of xe2x80x94NRCOCONu, xe2x80x94NHCOCR2CR2CONu, xe2x80x94NHCOCRxe2x95x90CRCONu, and xe2x80x94NHCOSSCONu, wherein each R independently represents H or a substituted or unsubstituted alkyl group, and Nu represents a nucleophile.
The present invention will now be discussed by way of example. The following examples are meant to be illustrative not limiting.